ACTH stimulation of steroidogenesis is thought to be mediated by cyclic AMP. ACTH treatment promotes increased cyclic AMP synthesis prior to stimulation of steroid synthesis; cyclic AMP stimulation initiates adrenal steroidogenesis. Recent studies indicate that cyclic AMP stimulates steroidogenesis by activating protein kinases. The protein kinases increase the phosphorylation of various adrenal proteins which mediate the steroidogenic effect. This proposal is designed to investigate the relationship between protein phosphorylation and stimulation of steroidogenesis in steroid secreting adrenal tumor cells and in isolated rat adrenal cells. The functional relationship between phosphorylation and steroidogenesis has not been investigated. Although evidence suggests that phosphorylation may be required for stimulation of steroidogenesis, the requirement has not been tested. The need to obtain evidence of the involvement of phosphorylation has become particularly apparent from our studies indicating that the adrenal cyclic AMP second-messenger-hypothesis may require drastic revision. The requirement for phosphorylation will be studied by developing an assay for measurement of phosphoprotein turnover in intact adrenal cells using P33-phosphate and P32-phosphate tracers and pulse labelling techniques. The effects of ACTH and NPS-ACTH on this turnover in adrenal tumor and rat adrenal cells will be examined and correlated with cyclic AMP accumulation and steroidogenesis. NPS-ACTH is an ACTH derivative capable of stimulating steroidogenesis by itself ad inhibiting cyclic AMP accumulation induced by ACTH. In addition the effects of cyclic AMP and other nucleotides capable of stimulating steroidogenesis in the adrenal tumor cells on phosphoprotein turnover will be examined to determine whether all agents capable of enhancing steroidogenesis act similarly. These studies will indicate whether the ACTH and cyclic AMP stimulation of steroidogenesis is mediated by phosphoproteins as has been postulated on the basis of studies in isolated enzyme systems.